Hepatitis C virus (HCV) is a major cause of chronic liver disease. We demonstrated that HCV could be classified into 6 major genotypes. Prototype strains of the various genotypes of HCV have been biologically amplified in chimpanzees and distributed for use as challenge inocula in studies of passive and active immunoprophylaxis, etc. Full-length cDNA clones of genotypes 1a, 1b and 2a have been constructed and transcribed RNA used to transmit hepatitis C to chimpanzees by in vivo hepatic transfection. With the reagents we have developed we are pursuing studies of the immunopathogenesis of HCV infections in chimpanzees, a surrogate of man. Humoral immunity. We have shown that humoral immunity is not important in the control of acute infection, since antibodies against the viral envelope proteins (E1 and E2), as measured against recombinant genotype 1a proteins, and neutralizing antibodies, as measured against pseudotyped retroviruses bearing the HCV genotype 1a glycoproteins, was detected only in 1 of 4 animals with acute resolving 1a infection. High titer neutralizing antibodies were detected in 2 of 4 chimpanzees with persistent infection; responses correlated with humoral responses to the E1 and E2 proteins. Similarly, high titers of neutralizing antibodies were detected during persistent genotype 1a infection in a patient with posttransfusion hepatitis. We have developed infectious pseudo-typed particles bearing also the envelope proteins of genotypes 2a, 3a, 4a, and 5a and demonstrated that neutralizing antibodies that develop during persistent 1a infection can cross-neutralize pseudo-particles of genotypes 4a and 5a, but had limited cross-reactivity against pseudo-particles of genotypes 2a and 3a. Cellular immunity. We have demonstrated that the intrahepatic CD4+ and CD8+ T cell responses were vigorous in chimpanzees with viral control but their strength does not necessarily predict the final outcome of infection. Since animals infected with the same HCV strain had mild to severe acute liver disease these studies also suggested that the host response is the principal determinant of the severity of acute HCV. Host gene expression. Gene expression analysis (RNase protection assay and microarray) of HCV infected chimpanzees with different outcomes identified intrahepatic responses associated with HCV viremia, such as interferon alpha, as well as outcome-specific responses associated with clearance, such as genes involved in the adaptive immune response. These studies also showed that acute HCV infection influences the expression of a large number of other genes, including those involved with metabolism, apoptosis and cell cycle regulation. Virus evolution. We have studied the correlation between host response, virus evolution and outcome in chimpanzees infected with different HCV strains. Changes in the polyprotein sequence are not selected until after the initial decrease in virus titers and until after the development of cellular immune responses and hepatitis. Subsequently mutations emerge repeatedly suggesting that mutations might represent an important mechanism for HCV persistence. However, the emergence of such variants does not necessarily lead to viral persistence. We have demonstrated that mutations that develop in animals with a monoclonal infection in some instances do represent CTL escape mutants. However, we have also demonstrated immune escape from the humoral response against the envelope proteins, in particular within HVR1. Protective immunity. We have demonstrated that sterilizing immunity can be achieved by repeated infection of chimpanzees, but that this immunity is strain-specific. This immunity is not mediated by neutralizing antibodies, but is correlated with anamnestic T cell responses. However, immunity acquired during acute HCV infection does not necessarily prevent persistent infection, even following rechallenge with the homologous monoclonal virus. Our analyses suggest that the persisting viruses represent immune escape variants. In vivo functional analysis. The availability of infectious cDNA clones has permitted mutational analysis of HCV. Infectivity of RNA transcripts of altered clones can be tested by inoculation of chimpanzees by intrahapatic transfection. We demonstrated that several regions of the 3' UTR were critical for in vivo replication of HCV. In other studies we found that the E2 HVR1 is not critical for viability of HCV and that this region is not essential for the resolution of infection or the progression to chronicity since both outcomes were observed. Mutational analysis of the gene that encodes the p7 protein demonstrated that p7 is critical for the viability of HCV and that this protein contains critical genotype-specific sequences. Finally, we have determined the in vivo effect of mutations that permit replication of a subgenomic replicon derived from the HCV strain Con1 in Huh-7 cells. The level of replication of replicons, as well as full-length Con1 genomes, in Huh-7 cells increased significantly by introducing adaptive mutations in NS3 and NS5A. However, these cell culture-adaptive mutations negatively influenced in vivo infectivity. Vaccine development. A DNA vaccine expressing a truncated E2 protein generated high anti-E2 titers in chimpanzees, but it did not prevent infection following homologous monoclonal challenge. It might have influenced the outcome of the infection since both vaccinated chimpanzees had early viral clearance. Recent studies have demonstrated that neither animal had detectable neutralizing antibodies against the homologous HCV pseudo-particles, which might explain why these animals were not fully protected. DNA vaccine vectors encoding other modified E2 proteins have been tested in rhesus monkeys. They did generate anti-E2 antibodies, but it remains to be determined whether they generated neutralizing antibodies. Similar studies of surface-expressed forms of E1 have been performed. These include E1 truncated at various sites and constructs with modification of glycosylation sites. The E1 constructs were tested as DNA vaccines in mice and a strategy of DNA vaccination followed by a vaccinia-mediated protein boost was explored. The vaccines generated E1 antibodies, but it remains to be determined whether they also generated neutralizing antibodies. Should any of the E1 or E2 vaccine candidates prove to generate reasonable titers of neutralizing antibodies, vaccination and challenge studies would be performed in chimpanzees. Using the pseudo-typed viruses bearing the envelope proteins of the various genotypes of HCV we have demonstrated cross-reactivity of monoclonal antibodies derived from humans or chimpanzees. Such antibodies could potentially be used in passive immnunoprophylaxis. A surrogate model for HCV. We have constructed an infectious cDNA clone of GB virus B (GBV-B), a monkey virus that is the closest relative to HCV. Experimental infection with GBV-B results in acute viral hepatitis in tamarins. By testing deletion mutants of the GBV-B clone in tamarins we have shown that each of the predicted encoded GBV-B proteins is critical for the virus. However, viruses with deletions of specific domains within the 3' UTR were found to be viable. Infection with one such deletion mutant produced a persistent infection with chronic hepatitis. This observation strengthens the relevance of GBV-B as a surrogate model for the study of HCV. Most recently, we have demonstrated that a p7 like protein of GBV-B exists and that this protein most likely is twice as large as the equivalent protein of HCV. We have demonstrated that this protein has two smaller subunits and that each subunit is essential for infectivity.